The present invention relates to a method and tools for extracting quantitative information relating to an influence, on a cellular response, in particular an influence caused by contacting or incubating the cell with a substance influencing a cellular response, where the cellular response is manifested in redistribution of at least one component in the cell. In particular, the invention relates to a method for extracting quantitative information relating to an influence on an intracellular pathway involving redistribution of at least one component associated with the pathway. The method of the invention may be used as a very efficient procedure for testing or discovering the influence of a substance on a physiological process, for example in connection with screening for new drugs, testing of substances for toxicity, identifying drug targets for known or novel drugs. Other valuable uses of the method and technology of the invention will be apparent to the skilled person on the basis of the following disclosure. In a particular embodiment of the invention, the present invention relates to a method of detecting intracellular translocation or redistribution of biologically active polypeptides, preferably an enzyme, affecting intracellular processes, and a DNA construct and a cell for use in the method.
Intracellular pathways are tightly regulated by a cascade of components that undergo modulation in a temporally and spatially characteristic manner. Several disease states can be attributed to altered activity of individual signalling components (i.e. protein kinases, protein phosphatases, transcription factors). These components therefore render themselves as attractive targets for therapeutic intervention.
Protein kinases and phosphatases are well described components of several intracellular signalling pathways. The catalytic activity of protein kinases and phosphatases are assumed to play a role in virtually all regulatable cellular processes. Although the involvement of protein kinases in cellular signalling and regulation have been subjected to extensive studies, detailed knowledge on e.g. the exact timing and spatial characteristics of signalling events is often difficult to obtain due to lack of a convenient technology.
Novel ways of monitoring specific modulation of intracellular pathways in intact, living cells is assumed to provide new opportunities in drug discovery, functional genomics, toxicology, patient monitoring etc.
The spatial orchestration of protein kinase activity is likely to be essential for the high degree of specificity of individual protein kinases. The phosphorylation mediated by protein kinases is balanced by phosphatase activity. Also within the family of phosphatases translocation has been observed, e.g. translocation of PTP2C to membrane ruffles [(Cossette et al. 1996)], and likewise is likely to be indicative of phosphatase activity.
Protein kinases often show a specific intracellular distribution before, during and after activation. Monitoring the translocation processes and/or redistribution of individual protein kinases or subunits thereof is thus likely to be indicative of their functional activity. A connection between translocation and catalytic activation has been shown for protein kinases like the diacyl glycerol (DAG)-dependent protein kinase C (PKC), the cAMP-dependent protein kinase (PKA) [(DeBernardi et al. 1996)] and the mitogen-activated-protein kinase Erk-1 [(Sano et al. 1995)].
Commonly used methods of detection of intracellular localisation/activity of protein kinases and phosphatases are immunoprecipitation, Western blotting and immunocytochemical detection.
Taking the family of diacyl glycerol (DAG)-dependent protein kinase Cs (PKCs) as an example, it has been shown that individual PKC isoforms that are distributed among different tissues and cells have different activator requirements and undergo differential translocation in response to activation. Catalytically inactive DAG-dependent PKCs are generally distributed throughout the cytoplasm, whereas they upon activation translocate to become associated with different cellular components, e.g. plasma membrane [(Farese, 1992),(Fulop Jr. et al. 1995)] nucleus [(Khalil et al. 1992)], cytoskeleton [(Blobe et al. 1996)]. The translocation phenomenon being indicative of PKC activation has been monitored using different approaches: a) immunocytochemistry where the localisation of individual isoforms can be detected after permeabilisation and fixation of the cells [(Khalil et al. 1992)]; and b) tagging all DAG-dependent PKC isoforms with a fluorescently labelled phorbol myristate acetate (PMA) [(Godson et al. 1996)]; and c) chemical tagging PKC b1 with the fluorophore Cy3 [(Bastiaens and Jovin 1996)] and d) genetic tagging of PKCxcex1 ([Schmidt et al. 1997]) and of PKCxcex3 and PKC xcex5 ([Sakai et at 1996]). The first method does not provide dynamic information whereas the latter methods will. Tagging PKC with fluorescently labelled phorbol myristate acetate cannot distinguish between different DAG-dependent isoforms of PKC but will label and show movement of all isoforms. Chemical and genetic labelling of specific DAG-dependent PKCs confirmed that they in an isoform specific manner upon activation move to cell periphery or nucleus.
In an alternative method, protein kinase A activity has been measured in living cells by chemical labelling one of the kinase""s subunit (Adams et al. 1991). The basis of the methodology is that the regulatory and catalytic subunit of purified protein kinase A is labelled with fluorescein and rhodamine, respectively. At low cAMP levels protein kinase A is assembled in a heterotetrameric form which enables fluorescence resonance energy transfer between the two fluorescent dyes. Activation of protein kinase A leads to dissociation of the complex, thereby eliminating the energy transfer. A disadvantage of this technology is that the labelled protein kinase A has to be microinjected into the cells of interest. This highly invasive technique is cumbersome and not applicable to large scale screening of biologically active substances. A further disadvantage of this technique as compared to the presented invention is that the labelled protein kinase A cannot be inserted into organisms/animals as a transgene. Recently it was discovered that Green Fluorescent Protein (GFP) expressed in many different cell types, including mammalian cells, became highly fluorescent [(Chalfie et al. 1994)]. WO95/07463 describes a cell capable of expressing GFP and a method for detecting a protein of interest in a cell based on introducing into a cell a DNA molecule having DNA sequence encoding the protein of interest linked to DNA sequence encoding a GFP such that the protein produced by the DNA molecule will have the protein of interest fused to the GFP, then culturing the cells in conditions permitting expression of the fused protein and detecting the location of the fluorescence in the cell, thereby localizing the protein of interest in the cell. However, examples of such fused proteins are not provided, and the use of fusion proteins with GFP for detection or quantitation of translocation or redistribution of biologically active polypeptides affecting intracellular processes upon activation, such as proteins involved in signalling pathways, e.g. protein kinases or phosphatases, has not been suggested. WO 95/07463 further describes cells useful for the detection of molecules, such as hormones or heavy metals, in a biological sample, by operatively linking a regulatory element of the gene which is affected by the molecule of interest to a GFP, the presence of the molecules will affect the regulatory element which in turn will affect the expression of the GFP. In this way the gene encoding GFP is used as a reporter gene in a cell which is constructed for monitoring the presence of a specific molecular identity.
Green Fluorescent Protein has been used in an assay for the detection of translocation of the glucocorticoid receptor (GR) [Carey, K L et al., The Journal of Cell Biology, Vol. 133, No. 5, p. 985-996 (1996)]. A GR-S65TGFP fusion has been used to study the mechanisms involved in translocation of the glucocorticoid receptor (GR) in response to the agonist dexamethasone from the cytosol, where it is present in the absence of a ligand, through the nuclear pore to the nucleus where it remains after ligand binding. The use of a GR-GFP fusion enables real-time imaging and quantitation of nuclear/cytoplasmic ratios of the fluorescence signal.
Many currently used screening programmes designed to find compounds that affect protein kinase activity are based on measurements of kinase phosphorylation of artificial or natural substrates, receptor binding and/or reporter gene expression.
The present invention provides an important new dimension in the investigation of cellular systems involving redistribution in that the invention provides quantification of the redistribution responses or events caused by an influence, typically contact with a chemical substance or mixture of chemical substances, but also changes in the physical environment. The quantification makes it possible to set up meaningful relationships, expressed numerically, or as curves or graphs, between the influences (or the degree of influences) on cellular systems and the redistribution response. This is highly advantageous because, as has been found, the quantification can be achieved in both a fast and reproducible manner, andxe2x80x94what is perhaps even more importantxe2x80x94the systems which become quantifiable utilizing the method of the invention are systems from which enormous amounts of new information and insight can be derived.
The present screening assays have the distinct advantage over other screening assays, e.g., receptor binding assays, enzymatic assays, and reporter gene assays, in providing a system in which biologically active substances with completely novel modes of action, e.g. inhibition or promotion of redistribution/translocation of a biologically active polypeptide as a way of regulating its action rather than inhibition/activation of enzymatic activity, can be identified in a way that insures very high selectivity to the particular isoform of the biologically active polypeptide and further development of compound selectivity versus other isoforms of the same biologically active polypeptide or other components of the same signalling pathway.
In its broadest aspect, the invention relates to a method for extracting quantitative information relating to an influence on a cellular response, the method comprising recording variation, caused by the influence on a mechanically intact living cell or mechanically intact living cells, in spatially distributed light emitted from a luminophore, the luminophore being present in the cell or cells and being capable of being redistributed in a manner which is related with the degree of the influence, and/or of being modulated by a component which is capable of being redistributed in a manner which is related to the degree of the influence, the association resulting in a modulation of the luminescence characteristics of the luminophore, detecting and recording the spatially distributed light from the luminophore, and processing the recorded variation in the spatially distributed light to provide quantitative information correlating the spatial distribution or change in the spatial distribution to the degree of the influence. In a preferred embodiment of the invention the luminophore, which is present in the cell or cells, is capable of being redistributed by modulation of an intracellular pathway, in a manner which is related to the redistribution of at least one component of the intracellular pathway. In another preferred embodiment of the invention, the luminophore is a fluorophore.
The Cells
In the invention the cell and/or cells are mechanically intact and alive throughout the experiment. In another embodiment of the invention, the cell or cells is/are fixed at a point in time after the application of the influence at which the response has been predetermined to be significant, and the recording is made at an arbitrary later time.
The mechanically intact living cell or cells could be selected from the group consisting of fungal cell or cells, such as a yeast cell or cells; invertebrate cell or cells including insect cell or cells; and vertebrate cell or cells, such as mammalian cell or cells. This cell or these cells is/are incubated at a temperature of 30xc2x0 C. or above, preferably at a temperature of from 32xc2x0 C. to 39xc2x0 C., more preferably at a temperature of from 35xc2x0 C. to 38xc2x0 C., and most preferably at a temperature of about 37xc2x0 C. during the time period over which the influence is observed. In one aspect of the invention the mechanically intact living cell is part of a matrix of identical or non-identical cells.
A cell used in the present invention should contain a nucleic acid construct encoding a fusion polypeptide as defined herein and be capable of expressing the sequence encoded by the construct. The cell is a eukaryotic cell selected from the group consisting of fungal cells, such as yeast cells; invertebrate cells including insect cells; vertebrate cells such as mammalian cells. The preferred cells are mammalian cells.
In another aspect of the invention the cells could be from an organism carrying in at least one of its component cells a nucleic acid sequence encoding a fusion polypeptide as defined herein and be capable of expressing said nucleic acid sequence. The organism is selected from the group consisting of unicellular and multicellular organisms, such as a mammal.
The Luminophore
The luminophore is the component which allows the redistribution to be visualised and/or recorded by emitting light in a spatial distribution related to the degree of influence. In one embodiment of the invention, the luminophore is capable of being redistributed in a manner which is physiologically relevant to the degree of the influence. In another embodiment, the luminophore is capable of associating with a component which is capable of being redistributed in a manner which is physiologically relevant to the degree of the influence. In another embodiment, the luminophore correlation between the redistribution of the luminophore and the degree of the influence could be determined experimentally. In a preferred aspect of the invention, the luminophore is capable of being redistributed in substantially the same manner as the at least one component of an intracellular pathway. In yet another embodiment of the invention, the luminophore is capable of being quenched upon spatial association with a component which is redistributed by modulation of the pathway, the quenching being measured as a change in the intensity of the luminescence.
The luminophore could be a fluorophore. In a preferred embodiment of the invention, the luminophore could be a polypeptide encoded by and expressed from a nucleotide sequence harboured in the cell or cells. The luminophore could be a hybrid polypeptide comprising a fusion of at least a portion of each of two polypeptides one of which comprises a luminescent polypeptide and the other one of which comprises a biologically active polypeptide, as defined herein.
The luminescent polypeptide could be a GFP as defined herein or could be selected from the group consisting of green fluorescent proteins having the F64L mutation as defined herein such as F64L-GFP, F64L-Y66H-GFP, F64L-S65T-GFP, and EGFP. The GFP could be N- or C-terminally tagged, optionally via a peptide linker, to the biologically active polypeptide or a part or a subunit thereof. The fluorescent probe could be a component of a intracellular signalling pathway. The probe is coded for by a nucleic acid construct.
The pathway of investigation in the present invention could be an intracellular signalling pathway.
The Influence
In a preferred embodiment of the invention, the influence could be contact between the mechanically intact living cell or the group of mechanically intact living cells with a chemical substance and/or incubation of the mechanically intact living cell or the group of mechanically intact living cells with a chemical substance. The influence will modulate the intracellular processes. In one aspect the modulation could be an activation of the intracellular processes. In another aspect the modulation could be an deactivation of the intracellular processes. In yet another aspect, the influence could inhibit or promote the redistribution without directly affecting the metabolic activity of the component of the intracellular processes.
In one embodiment the invention is used as a basis for a screening program, where the effect of unknown influences such as a compound library, can be compared to influence of known reference compounds under standardised conditions.
The Recording
In addition to the intensity, there are several parameters of fluorescence or luminescence which can be modulated by the effect of the influence on the underlying cellular phenomena, and can therefore be used in the invention. Some examples are resonance energy transfer, fluorescence lifetime, polarisation, wavelength shift. Each of these methods requires a particular kind of filter in the emission light path to select the component of the light desired and reject other components. The recording of property of light could be in the form of an ordered array of values such as a CCD array or a vacuum tube device such as a vidicon tube.
In one embodiment of the invention, the spatially distributed light emitted by a luminophore could be detected by a change in the resonance energy transfer between the luminophore and another luminescent entity capable of delivering energy to the luminophore, each of which has been selected or engineered to become part of, bound to or associated with particular components of the intracellular pathway. In this embodiment, either the luminophore or the luminescent entity capable of delivering energy to the luminophore undergoes redistribution in response to an influence. The resonance energy transfer would be measured as a change in the intensity of emission from the luminophore, preferably sensed by a single channel photodetector which responds only to the average intensity of the luminophore in a non-spatially resolved fashion.
In one embodiment of the invention, the recording of the spatially distributed light could be made at a single point in time after the application of the influence. In another embodiment, the recording could be made at two points in time, one point being before, and the other point being after the application of the influence. The result or variation is determined from the change in fluorescence compared to the fluorescence measured prior to the influence or modulation. In another embodiment of the invention, the recording could be performed at a series of points in time, in which the application of the influence occurs at some time after the first time point in the series of recordings, the recording being performed, e.g., with a predetermined time spacing of from 0.1 seconds to 1 hour, preferably from 1 to 60 seconds, more preferably from 1 to 30 seconds, in particular from 1 to 10 seconds, over a time span of from 1 second to 12 hours, such as from 10 seconds to 12 hours, e.g., from 10 seconds to one hour, such as from 60 seconds to 30 minutes or 20 minutes. The result or variation is determined from the change in fluorescence over time. The result or variation could also be determined as a change in the spatial distribution of the fluorescence over time.
Apparatus
The recording of spatially distributed luminescence emitted from the luminophore is performed by an apparatus for measuring the distribution of fluorescence in the cell or cells, and thereby any change in the distribution of fluorescence in the cell or cells, which includes at a minimum the following component parts: (a) a light source, (b) a method for selecting the wavelength(s) of light from the source which will excite the fluorescence of the protein, (c) a device which can rapidly block or pass the excitation light into the rest of the system, (d) a series of optical elements for conveying the excitation light to the specimen, collecting the emitted fluorescence in a spatially resolved fashion, and forming an image from this fluorescence emission, (e) a bench or stand which holds the container of the cells being measured in a predetermined geometry with respect to the series of optical elements, (f) a detector to record the spatially resolved fluorescence in the form of an image, (g) a computer or electronic system and associated software to acquire and store the recorded images, and to compute the degree of redistribution from the recorded images.
In a preferred embodiment of the invention the apparatus system is automated. In one embodiment the components in d and e mentioned above comprise a fluorescence microscope.
In one embodiment the component in f mentioned above is a CCD camera.
In one embodiment the image is formed and recorded by an optical scanning system.
In one embodiment a liquid addition system is used to add a known or unknown compound to any or all of the cells in the cell holder at a time determined in advance. Preferably, the liquid addition system is under the control of the computer or electronic system. Such an automated system can be used for a screening program due to its ability to generate results from a larger number of test compounds than a human operator could generate using the apparatus in a manual fashion.
Quantitation of the Influence
The recording of the variation or result with respect to light emitted from the luminophore is performed by recording the spatially distributed light as one or more digital images, and the processing of the recorded variation to reduce it to one or more numbers representative of the degree of redistribution comprises a digital image processing procedure or combination of digital image processing procedures. The quantitative information which is indicative of the degree of the cellular response to the influence or the result of the influence on the intracellular pathway is extracted from the recording or recordings according to a predetermined calibration based on responses or results, recorded in the same manner, to known degrees of a relevant specific influence. This calibration procedure is developed according to principles described below (Developing an Image-based Assay Technique). Specific descriptions of the procedures for particular assays are given in the examples.
While the stepwise procedure necessary to reduce the image or images to the value representative of the is particular to each assay, the individual steps are generally well-known methods of image processing. Some examples of the individual steps are point operations such as subtraction, ratioing, and thresholding, digital filtering methods such as smoothing, sharpening, and edge detection, spatial frequency methods such as Fourier filtering, image cross-correlation and image autocorrelation, object finding and classification (blob analysis), and colour space manipulations for visualisation. In addition to the algorithmic procedures, heuristic methods such as neural networks may also be used.
Nucleic Acid Constructs
The nucleic acid constructs used in the present invention encode in their nucleic acid sequences fusion polypeptides comprising a biologically active polypeptide that is a component of an intracellular signalling pathway, or a part thereof, and a GFP, preferably an F64L mutant of GFP, N- or C-terminally fused, optionally via a peptide linker, to the biologically active polypeptide or part thereof.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a protein kinase or a phosphatase.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a transcription factor or a part thereof which changes cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a protein, or a part thereof, which is associated with the cytoskeletal network and which changes cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a protein kinase or a part thereof which changes cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a serine/threonine protein kinase or a part thereof capable of changing intracellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a tyrosine protein kinase or a part thereof capable of changing intracellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a phospholipid-dependent serine/threonine protein kinase or a part thereof capable of changing intracellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a cAMP-dependent protein kinase or a part thereof capable of changing cellular localisation upon activation. In a preferred embodiment the biologically active polypeptide encoded by the nucleic acid construct is a PKAc-F64L-S65T-GFP fusion.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a cGMP-dependent protein kinase or a part thereof capable of changing cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a calmodulin-dependent serine/threonine protein kinase or a part thereof capable of changing cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a mitogen-activate serine/threonine protein kinase or a part thereof capable of changing cellular localisation upon activation. In preferred embodiments the biologically active polypeptide encoded by the nucleic acid constructs are an ERK1-F64L-S65T-GFP fusion or an EGFP-ERK1 fusion.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a cyclin-dependent serine/threonine protein kinase or a part thereof capable of changing cellular localisation upon activation.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct is a protein phosphatase or a part thereof capable of changing cellular localisation upon activation.
In one preferred embodiment of the invention the nucleic acid constructs may be DNA constructs.
In one embodiment the biologically active polypeptide encoded by the nucleic acid construct
In one embodiment the gene encoding GFP in the nucleic acid construct is derived from Aequorea victoria. In a preferred embodiment the gene encoding GFP in the nucleic acid construct is EGFP or a GFP variant selected from F64L-GFP, F64L-Y66H-GFP and F64L-S65T-GFP.
In preferred embodiments of the invention the DNA constructs which can be identified by any of the DNA sequences shown in SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142 or are variants of these sequences capable of encoding the same fusion polypeptide or a fusion polypeptide which is biologically equivalent thereto, e.g. an isoform, or a splice variant or a homologue from another species.
Screening Program
The present invention describes a method that may be used to establish a screening program for the identification of biologically active substances that directly or indirectly affects intracellular signalling pathways and because of this property are potentially useful as medicaments. Based on measurements in living cells of the redistribution of spatially resolved luminescence from luminophores which undergo a change in distribution upon activation or deactivation of an intracellular signalling pathway the result of the individual measurement of each substance being screened indicates its potential biological activity.
In one embodiment of the invention the screening program is used for the identification of a biologically toxic substance as defined herein that exerts its toxic effect by interfering with an intracellular signalling pathway. Based on measurements in living cells of the redistribution of spatially resolved luminescence from luminophores which undergo a change in distribution upon activation or deactivation of an intracellular signalling pathway the result of the individual measurement of each substance being screened indicates its potential biologically toxic activity. In one embodiment of a screening program a compound that modulates a component of an intracellular pathway as defined herein, can be found and the therapeutic amount of the compound estimated by a method according to the method of the invention. In a preferred embodiment the present invention leads to the discovery of a new way of treating a condition or disease related to the intracellular function of a biologically active polypeptide comprising administration to a patient suffering from said condition or disease of an effective amount of a compound which has been discovered by any method according to the invention. In another preferred embodiment of the invention a method is established for identification of a new drug target or several new drug targets among the group of biologically active polypeptides which are components of intracellular signalling pathways.
In another embodiment of the invention an individual treatment regimen is established for the selective treatment of a selected patient suffering from an ailment where the available medicaments used for treatment of the ailment are tested on a relevant primary cell or cells obtained from said patient from one or several tissues, using a method comprising transfecting the cell or cells with at least one DNA sequence encoding a fluorescent probe according to the invention, transferring the transfected cell or cells back the said patient, or culturing the cell or cells under conditions permitting the expression of said probes and exposing it to an array of the available medicaments, then comparing changes in fluorescence patterns or redistribution patterns of the fluorescent probes in the intact living cell or cells to detect the cellular response to the specific medicaments (obtaining a cellular action profile), then selecting one or more medicament or medicaments based on the desired activity and acceptable level of side effects and administering an effective amount of these medicaments to the selected patient.
Back-tracking of a Signal Transduction Pathway
The present invention describes a method that may be used to establish a screening program for back-tracking signal transduction pathways as defined herein. In one embodiment the screening program is used to establish more precisely at which level one or several compounds affect a specific signal transduction pathway by successively or in parallel testing the influence of the compound or compounds on the redistribution of spatially resolved luminescence from several of the luminophores which undergo a change in distribution upon activation or deactivation of the intracellular signalling pathway under study.
Construction and Testing of Probes
In general, a probe, i.e. a xe2x80x9cGeneXxe2x80x9d-GFP fusion or a GFP-xe2x80x9cGeneXxe2x80x9d fusion, is constructed using PCR with xe2x80x9cGeneXxe2x80x9d-specific primers followed by a cloning step to fuse xe2x80x9cGeneXxe2x80x9d in frame with GFP. The fusion may contain a short vector derived sequence between xe2x80x9cGeneXxe2x80x9d and GFP (e.g. part of a multiple cloning site region in the plasmid) resulting in a peptide linker between xe2x80x9cGeneXxe2x80x9d and GFP in the resulting fusion protein.
Detailed Stepwise Procedure:
Identifying the sequence of the gene. This is most readily done by searching a depository of genetic information, e.g. the GenBank Sequence Database, which is widely available and routinely used by molecular biologists. In the specific examples below the GenBank Accession number of the gene in question is provided.
Design of gene-specific primers. Inspection of the sequence of the gene allows design of gene-specific primers to be used in a PCR reaction. Typically, the top-strand primer encompasses the ATG start codon of the gene and the following ca. 20 nucleotides, while the bottom-strand primer encompasses the stop codon and the ca. 20 preceding nucleotides, if the gene is to be fused behind GFP, i.e. a GFP-xe2x80x9cGeneXxe2x80x9d fusion. If the gene is to be fused in front of GFP, i.e. a xe2x80x9cGeneXxe2x80x9d-GFP fusion, a stop codon must be avoided. Optionally, the full length sequence of GeneX may not be used in the fusion, but merely the part which localizes and redistributes like GeneX in response to a signal.
In addition to gene-specific sequences, the primers contain at least one recognition sequence for a restriction enzyme, to allow subsequent cloning of the PCR product. The sites are chosen so that they are unique in the PCR product and compatible with sites in the cloning vector. Furthermore, it may be necessary to include an exact number of nucleotides between the restriction enzyme site and the gene-specific sequence in order to establish the correct reading frame of the fusion gene and/or a translation initiation consensus sequence. Lastly, the primers always contain a few nucleotides in front of the restriction enzyme site to allow efficient digestion with the enzyme.
Identifying a source of the gene to be amplified. In order for a PCR reaction to produce a product with gene-specific primers, the gene-sequence must initially be present in the reaction, e.g. in the form of cDNA. Information in GenBank or the scientific literature will usually indicate in which tissue(s) the gene is expressed, and cDNA libraries from a great variety of tissues or cell types from various species are commercially available, e.g. from Clontech (Palo Alto), Stratagene (La Jolla) and Invitrogen (San Diego). Many genes are also available in cloned form from The American Type Tissue Collection (Virginia).
Optimizing the PCR reaction. Several factors are known to influence the efficiency and specificity of a PCR reaction, including the annealing temperature of the primers, the concentration of ions, notably Mg2+ and K+, present in the reaction, as well as pH of the reaction. If the result of a PCR reaction is deemed unsatisfactory, it might be because the parameters mentioned above are not optimal. Various annealing temperatures should be tested, e.g. in a PCR machine with a built-in temperature gradient, available from e.g. Stratagene (La Jolla), and/or various buffer compositions should be tried, e.g. the OptiPrime buffer system from Stratagene (La Jolla).
Cloning the PCR product. The vector into which the amplified gene product will be cloned and fused with GFP will already have been taken into consideration when the primers were designed. When choosing a vector, one should at least consider in which cell types the probe subsequently will be expressed, so that the promoter controlling expression of the probe is compatible with the cells. Most expression vectors also contain one or more selective markers, e.g. conferring resistance to a drug, which is a useful feature when one wants to make stable transfectants. The selective marker should also be compatible with the cells to be used.
The actual cloning of the PCR product should present no difficulty as it typically will be a one-step cloning of a fragment digested with two different restriction enzymes into a vector digested with the same two enzymes. If the cloning proves to be problematic, it may be because the restriction enzymes did not work well with the PCR fragment. In this case one could add longer extensions to the end of the primers to overcome a possible difficulty of digestion close to a fragment end, or one could introduce an intermediate cloning step not based on restriction enzyme digestion. Several companies offer systems for this approach, e.g. Invitrogen (San Diego) and Clontech (Palo Alto).
Once the gene has been cloned and, in the process, fused with the GFP gene, the resulting product, usually a plasmid, should be carefully checked to make sure it is as expected. The most exact test would be to obtain the nucleotide sequence of the fusion-gene.
Testing the Probe
Once a DNA construct for a probe has been generated, its functionality and usefulness may be tested by subjecting it to the following tests:
Transfecting it into cells capable of expressing the probe. The fluorescence of the cell is inspected soon after, typically the next day. At this point, two features of cellular fluorescence are noted: the intensity and the sub-cellular localization.
The intensity should usually be at least as strong as that of unfused GFP in the cells. If it is not, the sequence or quality of the probe-DNA might be faulty, and should be carefully checked.
The sub-cellular localization is an indication of whether the probe is likely to perform well. If it localizes as expected for the gene in question, e.g. is excluded from the nucleus, it can immediately go on to a functional test. If the probe is not localized soon after the transfection procedure, it may be because of overexpression at this point in time, as the cell typically will have taken of very many copies of the plasmid, and localization will occur in time, e.g. within a few weeks, as plasmid copy number and expression level decreases. If localization does not occur after prolonged time, it may be because the fusion to GFP has destroyed a localization function, e.g. masked a protein sequence essential for interaction with its normal cellular anchor-protein. In this case the opposite fusion might work, e.g. if GeneX-GFP does not work, GFP-GeneX might, as two different parts of GeneX will be affected by the proximity to GFP. If this does not work, the proximity of GFP at either end might be a problem, and it could be attempted to increase the distance by incorporating a longer linker between GeneX and GFP in the DNA construct.
If there is no prior knowledge of localization, and no localization is observed, it may be because the probe should not be localized at this point, because such is the nature of the protein fused to GFP. It should then be subjected to a functional test.
In a functional test, the cells expressing the probe are treated with at least one compound known to perturb, usually by activating, the signalling pathway on which the probe is expected to report by redistributing itself within the cell. If the redistribution is as expected, e.g. if prior knowledge tell that it should translocate from location X to location Y, it has passed the first critical test. In this case it can go on to further characterization and quantification of the response.
If it does not perform as expected, it may be because the cell lacks at least one component of the signalling pathway, e.g. a cell surface receptor, or there is species incompatibility, e.g. if the probe is modelled on sequence information of a human geneproduct, and the cell is of hamster origin. In both instances one should identify other cell types for the testing process where these potential problems would not apply.
If there is no prior knowledge about the pattern of redistribution, the analysis of the redistribution will have to be done in greater depth to identify what the essential and indicative features are, and when this is clear, it can go on to further characterization and quantification of the response. If no feature of redistribution can be identified, the problem might be as mentioned above, and the probe should be retested under more optimal cellular conditions.
If the probe does not perform under optimal cellular conditions it""s back to the drawing board.
Developing an Image-based Assay Technique
The process of developing an image-based redistribution assay begins with either the unplanned experimental observation that a redistribution phenomenon can be visualised, or the design of a probe specifically to follow a redistribution phenomenon already known to occur. In either event, the first and best exploratory technique is for a trained scientist or technician to observe the phenomenon. Even with the rapid advances in computing technology, the human eye-brain combination is still the most powerful pattern recognition system known, and requires no advance knowledge of the system in order to detect potentially interesting and useful patterns in raw data. This is especially if those data are presented in the form of images, which are the natural xe2x80x9cdata typexe2x80x9d for human visual processing. Because human visual processing operates most effectively in a relatively narrow frequency range, i.e., we cannot see either very fast or very slow changes in our visual field, it may be necessary to record the data and play it back with either time dilation or time compression.
Some luminescence phenomena cannot be seen directly by the human eye. Examples include polarization and fluorescence lifetime. However, with suitable filters or detectors, these signals can be recorded as images or sequences of images and displayed to the human in the fashion just described. In this way, patterns can be detected and the same methods can be applied.
Once the redistribution has been determined to be a reproducible phenomenon, one or more data sets are generated for the purpose of developing a procedure for extracting the quantitative information from the data. In parallel, the biological and optical conditions are determined which will give the best quality raw data for the assay. This can become an iterative process; it may be necessary to develop a quantitative procedure in order to assess the effect on the assay of manipulating the assay conditions.
The data sets are examined by a person or persons with knowledge of the biological phenomenon and skill in the application of image processing techniques. The goal of this exercise is to determine or at least propose a method which will reduce the image or sequence of images constituting the record of a xe2x80x9cresponsexe2x80x9d to a value corresponding to the degree of the response. Using either interactive image processing software or an image processing toolbox and a programming language, the method is encoded as a procedure or algorithm which takes the image or images as input and generates the degree of response (in any units) as its output. Some of the criteria for evaluating the validity of a particular procedure are:
Does the degree of the response vary in a biologically significant fashion, i.e., does it show the known or putative dependence on the concentration of the stimulating agent or condition?
Is the degree of response reproducible, i.e., does the same concentration or level of stimulating agent or condition give the same response with an acceptable variance?
Is the dynamic range of the response sufficient for the purpose of the assay? If not, can a change in the procedure or one of its parameters improve the dynamic range?
Does the procedure exhibit any clear xe2x80x9cpathologiesxe2x80x9d, i.e., does it give ridiculous values for the response if there are commonly occurring imperfections in the imaging process? Can these pathologies be eliminated, controlled, or accounted for?
Can the procedure deal with the normal variation in the number and/or size of cells in an image?
In some cases the method may be obvious; in others, a number of possible procedures may suggest themselves. Even if one method appears clearly superior to others, optimisation of parameters may be required. The various procedures are applied to the data set and the criteria suggested above are determined, or the single procedure is applied repeatedly with adjustment of the parameter or parameters until the most satisfactory combination of signal, noise, range, etc. are arrived at. This is equivalent to the calibration of any type of single-channel sensor.
The number of ways of extracting a single value from an image are extremely large, and thus an intelligent approach must be taken to the initial step of reducing this number to a small, finite number of possible procedures. This is not to say that the procedure arrived at is necessarily the best procedurexe2x80x94but a global search for the best procedure is simply out of the question due to the sheer number of possibilities involved.
Image-based assays are no different than other assay techniques in that their usefulness is characterised by parameters such as the specificity for the desired component of the sample, the dynamic range, the variance, the sensitivity, the concentration range over which the assay will work, and other such parameters. While it is not necessary to characterise each and every one of these before using the assay, they represent the only way to compare one assay with another.
Example: Developing a Quantitative Assay for GLUT4 Translocation
GLUT4 is a member of the class of glucose transporter molecules which are important in cellular glucose uptake. It is known to translocate to the plasma membrane under some conditions of stimulation of glucose uptake. The ability to visualize the glucose uptake response noninvasively, without actually measuring glucose uptake, would be a very useful assay for anyone looking for, for example, treatments for type II diabetes.
A CHO cell line which stably expressed the human insulin receptor was used as the basis for a new cell line which stably expressed a fusion between GLUT4 and GFP. This cell line was expected to show translocation of GLUT4 to the plasma membrane as visualized by the movement of the GFP. The translocation could definitely be seen in the form of the appearance of local increases in the fluorescence in regions of the plasma membrane which had a characteristic shape or pattern. This is shown in FIG. 12.
These objects became known as xe2x80x9csnirclesxe2x80x9d, and the phenomenon of their appearance as xe2x80x9csnirclingxe2x80x9d. In order to quantitate their appearance, a method had to be found to isolate them as objects in the image field, and then enumerate them, measure their area, or determine some parameter about them which correlated in a dose-dependent fashion with the concentration of insulin to which the cells had been exposed. In order to separate the snircles, a binarization procedure was applied in which one copy of the image smoothed with a relatively severe gaussian kernel (sigma=2.5) was subtracted from another copy to which only a relatively light gaussian smooth had been applied (sigma=0.5). The resultant image was rescaled to its min/max range, and an automatic threshold was applied to divide the image into two levels. The thresholded image contains a background of one value all found object with another value. The found objects were first filtered through a filter to remove objects far too large and far too small to be snircles. The remaining objects, which represent snircles and other artifacts from the image with approximately the same size and intensity characteristics as snircles, are passed into a classification procedure which has been previously trained with many images of snircles to recognize snircles and exclude the other artifacts. The result of this procedure is a binary image which shows only the found snircles to the degree to which the classification procedure can accurately identify them. The total area of the snircles is then summed and this value is the quantitative measure of the degree of snircling for that image.
Definitions
In the present specification and claims, the term xe2x80x9can influencexe2x80x9d covers any influence to which the cellular response comprises a redistribution. Thus, e.g., heating, cooling, high pressure, low pressure, humidifying, or drying are influences on the cellular response on which the resulting redistribution can be quantified, but as mentioned above, perhaps the most important influences are the influences of contacting or incubating the cell or cells with substances which are known or suspected to exert and influence on the cellular response involving a redistribution contribution. In another embodiment of the invention the influence could be substances from a compound drug library.
In the present context, the term xe2x80x9cgreen fluorescent proteinxe2x80x9d is intended to indicate a protein which, when expressed by a cell, emits fluorescence upon exposure to light of the correct excitation wavelength (cf. [(Chalfie et al. 1994)]). In the following, GFP in which one or more amino acids have been substituted, inserted or deleted is most often termed xe2x80x9cmodified GFPxe2x80x9d. xe2x80x9cGFPxe2x80x9d as used herein includes wild-type GFP derived from the jelly fish Aequorea victoria and modifications of GFP, such as the blue fluorescent variant of GFP disclosed by Heim et al. (1994). Proc.Natl.Acad.Sci. 91:12501, and other modifications that change the spectral properties of the GFP fluorescence, or modifications that exhibit increased fluorescence when expressed in cells at a temperature above about 30xc2x0 C. described in PCT/DK96/00051, published as WO 97/11094 on Mar. 27, 1997 and hereby incorporated by reference, and which comprises a fluorescent protein derived from Aequorea Green Fluorescent Protein (GFP) or any functional analogue thereof, wherein the amino acid in position 1 upstream from the chromophore has been mutated to provide an increase of fluorescence intensity when the fluorescent protein of the invention is expressed in cells. Preferred GFP variants are F64L-GFP, F64L-Y66H-GFP and F64L-S65T-GFP. An especially preferred variant of GFP for use in all the aspects of this invention is EGFP (DNA encoding EGFP which is a F64L-S65T variant with codons optimized for expression in mammalian cells is available from Clontech, Palo Alto, plasmids containing the EGFP DNA sequence, cf. GenBank Acc. Nos. U55762, U55763).
The term xe2x80x9cintracellular signalling pathwayxe2x80x9d and xe2x80x9csignal transduction pathwayxe2x80x9d are intended to indicate the coordinated intracellular processes whereby a living cell transduce an external or internal signal into cellular responses. Said signal transduction will involve an enzymatic reaction said enzymes include but are not limited to protein kinases, GTPases, ATPases, protein phosphatases, phospholipases. The cellular responses include but are not limited to gene transcription, secretion, proliferation, mechanical activity, metabolic activity, cell death.
The term xe2x80x9csecond messengerxe2x80x9d is used to indicate a low molecular weight component involved in the early events of intracellular signal transduction pathways.
The term xe2x80x9cluminophorexe2x80x9d is used to indicate a chemical substance which has the property of emitting light either inherently or upon stimulation with chemical or physical means. This includes but is not limited to fluorescence, bioluminescence, phosphorescence, chemiluminescence.
The term xe2x80x9cmechanically intact living cellxe2x80x9d is used to indicate a cell which is considered living according to standard criteria for that particular type of cell such as maintenance of normal membrane potential, energy metabolism, proliferative capability, and has not experienced any physically invasive treatment designed to introduce external substances into the cell such as microinjection.
The term xe2x80x9cphysiologically relevantxe2x80x9d, when applied to an experimentally determined redistribution of an intracellular component, as measured by a change in the luminescence properties or distribution, is used to indicate that said redistribution can be explained in terms of the underlying biological phenomenon which gives rise to the redistribution.
The terms xe2x80x9cimage processingxe2x80x9d and xe2x80x9cimage analysisxe2x80x9d are used to describe a large family of digital data analysis techniques or combination of such techniques which reduce ordered arrays of numbers (images) to quantitative information describing those ordered arrays of numbers. When said ordered arrays of numbers represent measured values from a physical process, the quantitative information derived is therefore a measure of the physical process.
The term xe2x80x9cfluorescent probexe2x80x9d is used to indicate a fluorescent fusion polypeptide comprising a GFP or any functional part thereof which is N- or C-terminally fused to a biologically active polypeptide as defined herein, optionally via a peptide linker consisting of one or more amino acid residues, where the size of the linker peptide in itself is not critical as long as the desired functionality of the fluorescent probe is maintained. A fluorescent probe according to the invention is expressed in a cell and basically mimics the physiological behaviour of the biologically active polypeptide moiety of the fusion polypeptide.
The term xe2x80x9cmammalian cellxe2x80x9d is intended to indicate any living cell of mammalian origin. The cell may be an established cell line, many of which are available from The American Type Culture Collection (ATCC, Virginia, USA) or a primary cell with a limited life span derived from a mammalian tissue, including tissues derived from a transgenic animal, or a newly established immortal cell line derived from a mammalian tissue including transgenic tissues, or a hybrid cell or cell line derived by fusing different cell types of mammalian origin e.g. hybridoma cell lines. The cells may optionally express one or more non-native gene products, e.g. receptors, enzymes, enzyme substrates, prior to or in addition to the fluorescent probe. Preferred cell lines include but are not limited to those of fibroblast origin, e.g. BHK, CHO, BALB, or of endothelial origin, e.g. HUVEC, BAE (bovine artery endothelial), CPAE (cow pulmonary artery endothelial) or of pancreatic origin, e.g. RIN, INS-1, MIN6, bTC3, aTC6, bTC6, HIT, or of hematopoietic origin, e.g. adipocyte origin, e.g. 3T3-L1, neuronal/neuroendocrine origin, e.g. AtT20, PC12, GH3, muscle origin, e.g. SKMC, A10, C2C12, renal origin, e.g. HEK 293, LLC-PK1.
The term xe2x80x9chybrid polypeptidexe2x80x9d is intended to indicate a polypeptide which is a fusion of at least a portion of each of two proteins, in this case at least a portion of the green fluorescent protein, and at least a portion of a catalytic and/or regulatory domain of a protein kinase. Furthermore a hybrid polypeptide is intended to indicate a fusion polypeptide comprising a GFP or at least a portion of the green fluorescent protein that contains a functional fluorophore, and at least a portion of a biologically active polypeptide as defined herein provided that said fusion is not the PKCxcex1-GFP, PKCxcex3-GFP, and PKCxcex5-GFP disclosed by Schmidt et al. and Sakai et al., respectively. Thus, GFP may be N- or C-terminally tagged to a biologically active polypeptide, optionally via a linker portion or linker peptide consisting of a sequence of one or more amino acids. The hybrid polypeptide or fusion polypeptide may act as a fluorescent probe in intact living cells carrying a DNA sequence encoding the hybrid polypeptide under conditions permitting expression of said hybrid polypeptide.
The term xe2x80x9ckinasexe2x80x9d is intended to indicate an enzyme that is capable of phosphorylating a cellular component.
The term xe2x80x9cprotein kinasexe2x80x9d is intended to indicate an enzyme that is capable of phosphorylating serine and/or threonine and/or tyrosine in peptides and/or proteins.
The term xe2x80x9cphosphatasexe2x80x9d is intended to indicate an enzyme that is capable of dephosphorylating phosphoserine and/or phosphothreonine and/or phosphotyrosine in peptides and/or proteins.
In the present context, the term xe2x80x9cbiologically active polypeptidexe2x80x9d is intended to indicate a polypeptide affecting intracellular processes upon activation, such as an enzyme which is active in intracellular processes or a portion thereof comprising a desired amino acid sequence which has a biological function or exerts a biological effect in a cellular system. In the polypeptide one or several aminoacids may have been deleted, inserted or replaced to alter its biological function, e.g. by rendering a catalytic site inactive. Preferably, the biologically active polypeptide is selected from the group consisting of proteins taking part in an intracellular signalling pathway, such as enzymes involved in the intracellular phosphorylation and dephosphorylation processes including kinases, protein kinases and phosphorylases as defined herein, but also proteins making up the cytoskeleton play important roles in intracellular signal transduction and are therefore included in the meaning of xe2x80x9cbiologically active polypeptidexe2x80x9d herein. More preferably, the biologically active polypeptide is a protein which according to its state as activated or non-activated changes localisation within the cell, preferably as an intermediary component in a signal transduction pathway. Included in this preferred group of biologically active polypeptides are cAMP dependent protein kinase A.
The term xe2x80x9ca substance having biological activityxe2x80x9d is intended to indicate any sample which has a biological function or exerts a biological effect in a cellular system. The sample may be a sample of a biological material such as a sample of a body fluid including blood, plasma, saliva, milk, urine, or a microbial or plant extract, an environmental sample containing pollutants including heavy metals or toxins, or it may be a sample containing a compound or mixture of compounds prepared by organic synthesis or genetic techniques.
The phrase xe2x80x9cany change in fluorescencexe2x80x9d means any change in absorption properties, such as wavelength and intensity, or any change in spectral properties of the emitted light, such as a change of wavelength, fluorescence lifetime, intensity or polarisation, or any change in the intracellular localisation of the fluorophore. It may thus be localised to a specific cellular component (e.g. organelle, membrane, cytoskeleton, molecular structure) or it may be evenly distributed throughout the cell or parts of the cell.
The phrase xe2x80x9cback-tracking of a signal transduction pathwayxe2x80x9d is intended to indicate.
The term xe2x80x9corganismxe2x80x9d as used herein indicates any unicellular or multicellular organism preferably originating from the animal kingdom including protozoans, but also organisms that are members of the plant kingdoms, such as algae, fungi, bryophytes, and vascular plants are included in this definition.
The term xe2x80x9cnucleic acidxe2x80x9d is intended to indicate any type of poly- or oligonucleic acid sequence, such as a DNA sequence, a cDNA sequence, or an RNA sequence.
The term xe2x80x9cbiologically equivalentxe2x80x9d as it relates to proteins is intended to mean that a first protein is equivalent to a second protein if the cellular functions of the two proteins may substitute for each other, e.g. if the two proteins are closely related isoforms encoded by different genes, if they are splicing variants, or allelic variants derived from the same gene, if they perform identical cellular functions in different cell types, or in different species. The term xe2x80x9cbiologically equivalentxe2x80x9d as it relates to DNA is intended to mean that a first DNA sequence encoding a polypeptide is equivalent to a second DNA sequence encoding a polypeptide if the functional proteins encoded by the two genes are biologically equivalent.
The phrase xe2x80x9cback-tracking of a signal transduction pathwayxe2x80x9d is intended to indicate a process for defining more precisely at what level a signal transduction pathway is affected, either by the influence of chemical compounds or a disease state in an organism. Consider a specific signal transduction pathway represented by the bioactive polypeptides A-B-C-D, with signal transduction from A towards D. When investigating all components of this signal transduction pathway compounds or disease states that influence the activity or redistribution of only D can be considered to act on C or downstream of C whereas compounds or disease states that influence the activity or redistribution of C and D, but not of A and B can be considered to act downstream of B.
The term xe2x80x9cfixed cellsxe2x80x9d is used to mean cells treated with a cytological fixative such as glutaraldehyde or formaldehyde, treatments which serve to chemically cross-link and stabilize soluble and insoluble proteins within the structure of the cell. Once in this state, such proteins cannot be lost from the structure of the now-dead cell.